Cysteine-containing peptides having antioxidant properties

ABSTRACT

Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I Milano  (R173C) and apoA-I Paris , (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/142,238, filed on May 8, 2002, now issued U.S. Pat. No. 7,217,785,which claims benefit of Application No. 60/289,944, which was filed onMay 9, 2001.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made during work partially supported by the U.S.Department of Energy under Contract No. DE-AC03-76SF00098. This work wasalso supported by NIH grant HL59483. The government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to human lipid metabolism, particularlyto HDL-related proteins, their mutations, and peptides designed based onthese mutations which have antioxidant properties beneficial in theregulation of cardiovascular disease (CVD), bone diseases and otherinflammatory related diseases.

2. Description of the Related Art

Cardiovascular disease (CVD) is the number one cause of death in Westernsocieties and its prevalence is increasing worldwide. One of thestrongest predictors of risk is the plasma concentration of high densitylipoprotein (HDL) which exhibits an inverse relationship to the risk(Gordon, T., et al., Am. J. Med. 62:707-714, 1997; Wilson, P. W. F., Am.J. Cardiol. 66:7A-10A, 1990). Despite the strong epidemiological datarelating increased plasma HDL to protection against CVD, a number ofrare inheritable traits have been described which result in low plasmaHDL concentrations but no increase in CVD. These inheritable traits are,in part, attributed to mutations in apolipoproteinA-I, the major proteincomponent of HDL (Assmann, G., et al, Circulation 87:[supplIII]:III-28-III-34, 1993).

ApolipoproteinA-I_(Milano) and apoA-I_(Paris) are examples of naturalvariants of apoA-I that manifest HDL deficiencies but there is noapparent CVD in affected subjects. See Weisgraber, K. H., et al., J.Clin. Invest. 66:901-907, 1980; Franceschini, G., et al., J. Clin.Invest. 66:892-900, 1980; Bruckert, E., et al., Atherosclerosis,128:121-128, 1997. Indeed, a recent clinical study showed that carriersof apoA-I_(Milano) exhibited normal intimal thickness of carotidarteries compared to age- and sex-matched controls; whereas,hypoalphalipoproteinemic individuals showed intimal thickening as judgedby B-mode ultrasound (Sitori, C. R., et al., Circulation 103:1949-1954,2001). Studies utilizing mice and rabbits support clinical studies bydemonstrating that injection of recombinant apoA-I_(Milano) protectsagainst atherosclerosis (Shah, P. K., et al., Circulation 97:780-785,1998; Shah, P. K., et al., Circulation 103:3047-3050, 2001; Ameli, S.,et al., Circulation 90:1935-1941, 1994). However, the mechanism(s) bywhich apoA-I_(Milano) and apoA-I_(Paris) exert anti-atherogenic effectsare not completely understood.

All known human carriers of apoA-I_(Milano) and apoA-I_(Paris) areheterozygous for R173C and R151C mutations in apoA-I primary sequence,respectively (Weisgraber, K. H., et al., J. Clin. Invest. 66:901-907,1980; Bruckert, E., et al., Atherosclerosis, 128:121-128, 1997). Theintroduction of a cysteine residue in a normally cysteine-freeapolipoprotein allows for the formation of homodimers and heterodimerswith apoA-II. Dimerization of the cysteine variants inhibits HDLmaturation via mechanisms related, in part, to impaired activation oflecithin: cholesterol acyltransferase, the enzyme that catalyzescholesterol esterification on HDL (Franceschini, G., et al. J. BiolChem. 265:12224-12231, 1990; Calabresi, L., et al., Biochem. Biophys.Res. Comm. 232:345-349, 1997; Daum, U., et al., J. Mol. Med. 77:614-622,1999). ApoA-I_(Milano) and apoA-I_(Paris) are rapidly cleared from theplasma compartment in humans thus contributing to the HDL deficiency invivo (Roma P, et al., J. Clin. Invest. 91:1445-1452, 1993; Perez-Mendez,O., et al., Atherosclerosis 148:317-326, 2000). However, the fractionalcatabolic rate of apoA-I_(Paris) appears to be different from that ofapoA-I_(Milano) suggesting that the two cysteine variants may differ intheir metabolic behavior. Human carriers of apoA-I_(Milano) andapoA-I_(Paris) also exhibit mild hypertriglyceridemia in addition to theHDL deficiency (Bruckert, E., et al., Atherosclerosis, 128:121-128,1997; Franceschini G., et al., Atherosclerosis 7:426-435, 1987).

The C-terminal lipid-binding domain of ApoA-I_(WT) consists of a seriesof helical repeats separated by proline residues. The amphipathic alphahelix (a.a. 167-184) containing R173C is flanked by two amphipathicalpha helices of relatively greater lipid binding affinity. The lipidbinding affinity of the helical repeats alternate, but the two endhelices of apoA-I exhibit the highest lipid-binding affinity(Palgunachari, M. N., et al., Arterioscler. Thromb. Vasc. Biol.16:328-338, 1996). The relatively low lipid-binding affinity associatedwith helix 7, where R173C is located, may allow a high degree ofmovement of this particular helix on phospholipid surfaces thusmaximizing the frequency of collision between the free thiol at position173 with reactive lipid peroxides. Increased flexibility of helix 7,which is located in the central region of the C-terminal lipid-bindingdomain, may be optimized in the presence of deoxycholate used in thepreparation of the phospholipid micelles.

The paradox of abnormal lipoprotein metabolism and protection from CVDhas led to the suggestion that the cysteine substitution for arginine inthe lipid-binding domain of apoA-I may impart a gain-of-functionprotecting against atherosclerosis. As thiol groups in proteins arestrong nucleophiles often participating in electron transfer reactions,we hypothesized that the monomeric forms of apoA-I_(Milano) andapoA-I_(Paris), which contain a free thiol, may possess an antioxidantactivity distinct from that of apoA-I_(WT).

Individuals with these substitutions are known to have low levels of the“good” cholesterol HDL, but yet do not suffer from significantlyincreased levels of CVD. Oda et al. disclose cysteine substitutions inApolipoprotein A-I in Biochemistry 40 (2001) 1710-1718; othersubstitutions are disclosed at Atherosclerosis 128 (1997) 121-128;Atherosclerosis 135 (1997) 181-185. Antioxidant action of HDL isdiscussed at Atherosclerosis 135 (1997) 193-204.

These cysteine for arginine substitutions in the Apo A-I variant is ofspecial interest in treatment of cardiovascular disease. The dimer ofApolipoprotein A-I_(Milano) and the process of producing and purifyingthe dimer composition have been disclosed by Sirtori et al, in U.S. Pat.No. 5,876,968, which is hereby incorporated by reference. The processdescribed by Sirtori et al. relies on converting any monomer present toa substantially pure form of the dimer form of ApoA-I_(Milano) of atleast 90% purity.

Segrest et al., in U.S. Pat. No. 4,643,988, discloses amphipathicpeptides that are useful for treatment and prevention of athersclerosis.The Segrest peptides, generally referred to as 18A and 18pA, are basedon an idealistic model of an amphipathic alpha helix that possesses aprimary amino acid sequence distinct from that of apoA-I. However, thepeptides form Class A amphipathic alpha helices with positively chargedamino acids at the interface of polar/nonpolar region and negativelycharged residues located in the middle of the polar face of the helix.

Segrest et al. describe the use and properties of 18A and 18pA; thelatter representing a series of two 18A peptides linked by a prolineresidue. The sequence of 18A is as follows: DWLKAFYDKVAEKLKEAF (SEQ IDNO:75). Various conservative substitutions (for example positivelycharged lysine residues in place of positively charged arginineresidues) that do not change the overall design of the class Aamphipathic alpha helix are also claimed. Additional substitutions of D-for L-amino acid isoforms are described as well as replacement ofnaturally occurring amino acids for synthetic derivatives (i.e.substitutions of alanine for alpha-naphthylalanine). While anamphipathic peptide is disclosed, the peptide does not possess acysteine residue and thus lacks the antioxidant activity shown to bepossessed by apoA-I_(Milano).

Garber et al., disclose on U.S. Pat. No. 6,156,727, anti-atheroscleroticpeptides and a transgenic mouse model of atherosclerosis Garber et al.utilize the same peptides as described above by Segrest et al., howeverGarber et al. created transgenic mice that express the peptides 18A and37pA, the latter sometimes is referred to as 18A-Pro-18A. Again, thesepeptide does not possess a cysteine residue and thus lack theantioxidant activity shown to be possessed by apoA-I_(Milano)

Lees et al., disclose in U.S. Pat. No. 5,955,055, synthetic peptides forarterial imaging at vascular imaging sites, that mimic apolipoprotein B(apoB), apolipoprotein A-I or elastin proteins and is herebyincorporated by reference in its entirety. The Lees peptides are derived(mostly) from apoB and elastin/collagen and are not similar to thepeptides we now disclose. The following sequence is used as is based onapoB: YRALVDTLKFVTQAEGAL (SEQ ID NO:89). The sequence derived fromapoA-I described by Lees et al. is: YVLDEFREKLNEELEALKQ (SEQ ID NO:90).There is no exact sequence match to apoA-I, probably because ofconservative substitutions, and the peptide is not at all similar to anyof the peptides we now disclose.

Moreover, none of the peptides in the above mentioned patents are basedon Apolipoprotein E3 (apoE3)-and Apolipoprotein A-V (apoAV). This isbecause the mechanisms responsible for the antioxidant, properties ofapoE3 have not been fully defined until now. ApoAV is a newapolipoprotein that has recently been described and very little is knownabout its function. Thus, peptides based on apoAV provide new avenuesfor development of therapeutic agents. It is also clear from our studiesthat the antioxidant properties of apoA-I_(Milano) and its peptidemimetic-s are specifically directed toward phospholipid surfaces whichnone of these above-mentioned patented peptides are shown to be directedtoward.

BRIEF SUMMARY OF THE INVENTION

The present invention describes a new series of diagnostic andtherapeutic peptides that possess a novel antioxidant activity, such ashas been associated with the monomeric forms of apoA-I_(Milano) andapoA-I_(Paris) proteins. A critical feature of the present peptides isthe placement of a cysteine residue at the polar/nonpolar interface ofan amphipathic alpha helix just as in the apoA-I_(Milano) andapoA-I_(Paris) cysteine variants. The presence of a cysteine residue atthe polar/nonploar interface of the synthetic peptides confers a potentantioxidant activity that is directed toward lipid surfaces effectivelyblocking oxidation of phospholipid. The water accessibility of the freecysteine residue enables potential interaction with water-solubleantioxidants such as reduced glutathione thereby enhancing the overallcapacity of the peptides to inhibit phospholipid oxidation. Thisindicates that the peptides may be used in combination with other safeand effective therapies to promote beneficial interactions for long-termprotection against inflammatory related events. Structural analysesrevealed identical placement of a cysteine residue at the polar/nonpolarinterface of an amphipathic alpha helix within apoE3 thus defining themechanism for the antioxidant activity of apoE-III. A similar “motif” inapoAV is also used to create new peptides.

In the present peptides have also been developed in which the positionof the cysteine residue is changed around the face of the amphipathicalpha helix. Such changes in placement of the cysteine residue ispredicted to specifically alter the functionality of the peptides in asystematic fashion. For example, the cysteine residue is placed in themiddle of the nonploar face of the amphipathic alpha helix to inhibitspecific interaction with water-soluble antioxidants such as reducedglutathione. This enables the development of important biological toolsto determine whether such interactions are important in protectingagainst disease thus allowing the identification of new drug targets andproviding a basis for rationale drug design. This has led to thecreation of a generic amphipathic alpha helix for the development oftailor-made pharmacteuticals of defined functionality including specificantioxidant activity attributed to strategic cysteine placement, LCATactivation properties endowed via arginine clustering at thepolar/nonpolar interface, and cholesterol efflux properties obtained byeither phenylalanine placement or by combining unique helical segments.

The present invention comprises peptides possessing anti-oxidantactivity and which may be derived from naturally occurringHDL-associated proteins or may be designed de novo according to theprinciples outlined herein. The peptides of the present invention may becharacterized as follows, where the conventional single letter aminoacid code letters are used:

SEQ ID NO: 1 SDELRQCLAARLEALKEN 167-R173C-184 SEQ ID NO: 2SDELRQRLAARLEALKEN Control wild type 167-184 SEQ ID NO: 3SDELCQRLAARLEALKEN 167-R171C-184 SEQ ID NO: 4 SDELRCRLAARLEALKEN167-Q172C-184 SEQ ID NO: 5 SDELRQRCAARLEALKEN 167-L174C-184 SEQ ID NO: 6SDELRQRLCARLEALKEN 167-R175C-184 SEQ ID NO: 7 SDELRQRLACRLEALKEN167-A176C-184 SEQ ID NO: 8 SDELRQRLAARLEACKEN 167-L181C-184 SEQ ID NO: 9GEEMRDCARAHVDALRTH 145-R151C-162 SEQ ID NO: 10 GEEMRDRARAHVDALRTHControl wild type 145-162 SEQ ID NO: 11 GEEMCDRARAHVDALRTH 145-R149C-162SEQ ID NO: 12 GEEMRCRARAHVDALRTH 145-D150C-162 SEQ ID NO: 13GEEMRDRCRAHVDALRTH 145-A152C-162 SEQ ID NO: 14 GEEMRDRACAHVDALRTH145-R153C-162 SEQ ID NO: 15 GEEMRDRARACVDALRTH 145-H155C-162 SEQ ID NO:16 PVLESFCVSFLSALEEYT 220-K226C-237 SEQ ID NO: 17 PVLESFKVSFLSALEEYTControl wild type 220-237 SEQ ID NO: 18 PVLCSFKVSFLSALEEYT 220-E223C-237SEQ ID NO: 19 PVLECFKVSFLSALEEYT 220-S224C-237 SEQ ID NO: 20PVLESCKVSFLSALEEYT 220-F225C-237 SEQ ID NO: 21 PVLESFKCSFLSALEEYT220-V227C-237 SEQ ID NO: 22 PVLESFKVCFLSALEEYT 220-S228C-237 SEQ ID NO:23 PVLESFKVSCLSALEEYT 220-F229C-237 SEQ ID NO: 24 PVLESFKVSFCSALEEYT220-L230C-237 SEQ ID NO: 25 PVLESFKVSFLCALEEYT 220-S231C-237 SEQ ID NO:26 PVLESFKVSFLSCLEEYT 220-A232C-237 SEQ ID NO: 27 PVLESFKVSFLSALCEYT220-E234C-237 SEQ ID NO: 28 PVLESFKVSFLSALECYT 220-E235C-237 SEQ ID NO:29 PVLESFKVSFLSALEECT 220-Y236C-237 SEQ ID NO: 30 PALEDLRQGLL PVLESFCVSFLSALEEYT KKLN SEQ ID NO: 31 PALEDLRQGLL PVLESFK VSFLSALEEYT KKLN SEQID NO: 32 LKLCDNWDSVTSTFSKLR 44-L47C-61 SEQ ID NO: 33 LKLLDNWDSVTSTFSKLRControl wild type 44-61 SEQ ID NO: 34 LCLLDNWDSVTSTFSKLR 44-K45C-61 SEQID NO: 35 LKCLDNWDSVTSTFSKLR 44-L46C-61 SEQ ID NO: 36 LKLLCNWDSVTSTFSKLR44-D48C-61 SEQ ID NO: 37 LKLLDCWDSVTSTFSKLR 44-N49C-61 SEQ ID NO: 38LKLLDNWDSVTSTFSCLR 44-K59C-61 SEQ ID NO: 39 PALEDLRQGLLP LKLCDN209/44-L47C-61 WDSVTSTFSKLR SEQ ID NO: 40 PALEDLRQGLLP LKLLDNControl209/ 44-61 WDSVTSTFSKLR SEQ ID NO: 41 PALEDLCQGLLP LKLLDN209-R215C-220/ WDSVTSTFSKLR 44-61 SEQ ID NO: 42 PALEDLRQGLLP LCLLDN209/44-K45C-61 WDSVTSTFSKLR SEQ ID NO: 43 PALEDLRQGLLP LKCLDN209/44-L46C-61 WDSVTSTFSKLR SEQ ID NO: 44 PALEDLRQGLLP LKLLCN209/44-D48C-61 WDSVTSTFSKLR SEQ ID NO: 45 PALEDLRQGLLP LKLLDC209/44-N49C-61 WDSVTSTFSKLR SEQ ID NO: 46 PALEDLRQGLLP LKLLDN209/44--K59C-61 WDSVTSTFSCLR SEQ ID NO: 47 GADMEDVCGRLVQYRGEV105-R112C-122 SEQ ID NO: 48 GADMEDVRGRLVQYRGEV Control wild type 105-122SEQ ID NO: 49 GADMEDCRGRLVQYRGEV 105-V111C-122 SEQ ID NO: 50GADMEDVRCRLVQYRGEV 105-G113C-122 SEQ ID NO: 51 GADMEDVRGCLVQYRGEV105-R114C-122 SEQ ID NO: 52 ARLSRCVQVLSRKLTLKA 219-G224C-236 SEQ ID NO:53 ARLSRGVQVLSRKLTLKA Control wild type 219-236 SEQ ID NO: 54ARLCRGVQVLSRKLTLKA 219-S222C-236 SEQ ID NO: 55 ARLSCGVQVLSRKLTLKA219-R223C-236 SEQ ID NO: 56 ARLSRGCQVLSRKLTLKA 219-V225C-236 SEQ ID NO:57 ARLSRGVQVLSRKCTLKA 219-L232C-236 SEQ ID NO: 58 ARLSRCVQVLSRKLTLKAK219-G224C-254 ALHARIQQNLDQLREEL SEQ ID NO: 59 ARLSRGVQVLSRKLTLKAKControl 219-254 ALHARIQQNLDQLREEL SEQ ID NO: 60 ARLCRGVQVLSRKLTLKAK219-S222C-254 ALHARIQQNLDQLREEL SEQ ID NO: 61 ARLSCGVQVLSRKLTLKAK219-R223C-254 ALHARIQQNLDQLREEL SEQ ID NO: 62 ARLSRGCQVLSRKLTLKAK219-V225C-254 ALHARIQQNLDQLREEL SEQ ID NO: 63 ARLSRGVQVLSRKCTLKAK219-L232C-254 ALHARIQQNLDQLREEL SEQ ID NO: 64 ATLKDSLCQDLNNMNKFLE51-E58C-72 KLR SEQ ID NO: 65 ATLKDSLEQDLNNMNKFLE Control wild type KLR51-72 SEQ ID NO: 66 ATLCDSLEQDLNNMNKFLE 51-K54C-72 KLR SEQ ID NO: 67ATLKDCLEQDLNNMNKFLE 51-S56C-72 KLR SEQ ID NO: 68 ATLKDSCEQDLNNMNKFLE51-L57C-72 KLR SEQ ID NO: 69 ATLKDSLECDLNNMNKFLE 51-Q59C-72 KLR SEQ IDNO: 70 ATLKDSLEQCLNNMNKFLE 51-D60C-72 KLR SEQ ID NO: 71 ETGDLWVGCHP SEQID NO: 72 ETGDLWVGCHPNGMKIFFY DSEN SEQ ID NO: 73 LKSLDFNTLVDNISVDP ETGDLWVGCHPNGMKIFFYD SEN SEQ ID NO: 74 DWLCAFYDKVAEKLKEAF 18A-K4C SEQ IDNO: 75 DWLKAFYDKVAEKLKEAF 18A control SEQ ID NO: 76 DCLKAFYDKVAEKLKEAF18A-W2C SEQ ID NO: 77 DWCKAFYDKVAEKLKEAF 18A-L3C SEQ ID NO: 78DWLKCFYDKVAEKLKEAF 18A-A5C SEQ ID NO: 79 DWLKACYDKVAEKLKEAF 18A-F6C SEQID NO: 80 DWLKAFYDKCAEKLKEAF 18A-V10C SEQ ID NO: 81 DWLKAFYDKVCEKLKEAF18A-A11C SEQ ID NO: 82 LEKLNSCLRDRLSALTDTP LEELRDSLRSRLDALRST SEQ ID NO:83 LEKLNSCLRDRLSALTDT SEQ ID NO: 84 LEELRDSLRSRLDALRST

Preferred peptides are selected from helix 1 (amino acids 44-65), helix6 (amino acids 145-162) and helix 10 (amino acids 209-238) of apoAI,helix 7 (amino acids 167-184) of apoAI, the helix spanning amino acids105-122 of apoE3, and amino acids 219-236 of apo AV.

Furthermore, the present invention comprises peptide homologues of thesequences listed above, designed according to the detailed descriptionprovided below. The sequences listed above may be modified up to 80%homology without losing the functionality described herein. Furthermore,the sequences of the present invention may be provided with specificcysteine residues engineered into them. These cysteine residues may besubstitutes for the residues that are underlined in the sequences listedabove. That is, for example, SEQ ID NO: 2, SDELRQRLAARLEALKEN Controlwild type 167-184 has, according to the present invention at least onecysteine-residue in place of one of the underlined residues.

Furthermore, the present invention comprises methods for making ananti-oxidant peptide based on the design principles outlined in detailin the Detailed Description below. These methods include the steps ofidentifying an amphipathic helix by known methods for predictingsecondary structure and hydrophobicity. See Chou, P. Y., & Fasman, G.D., Adv. Enzymol. Relat. Areas Mol. Biol. 47, 45-148, 1978. A HumanHDL-associated protein of known amino acid sequence may be used for thispurpose. The identification of a helix as amphipathic is carried outusing conventional hydrophobicity analyses and helical wheelprojections. The alpha helices of the present invention will havebetween 10 and 100 amino acids, often between 8 and 30 amino acids.Being amphipathic, they will have a hydrophobic side and a hydrophilicside when viewed axially through the helix. As part of the design andsynthesis of the present peptides, one may modify at least one residueon the hydrophilic side from the naturally ocurring (wild type) aminoacid to a cysteine residue to create a modified helix peptide; and thenselecting a modified helix peptide that has at least twice theanti-oxidant activity as the unmodified peptide.

The Human HDL-associated protein may be selected from the groupconsisting of apoAI, apoE3, apo AV and paroxonase. Alternatively,synthetic or non-natural amino acids may be used in the presentpeptides.

The anti-oxidant activity is measured by the ability of the modifiedhelix peptide to inhibit lipid peroxidation by soybean lipoxygenase. Theability of the modified helix peptide to inhibit lipid peroxidation byxanthine oxidase and to (not) inhibit xanthine/xanthine oxidase mediatedreduction of cytochrome C may also be used to characterize the presentpeptides. These assays are described in detail in the DetailedDescription below.

In general, the present peptides as recited above will provideapproximately 50% or more protection against maximum accumulation oflipid peroxides at a concentration of no more than 500 micrograms permL. They will inhibit oxidation of a lipid or phospholipid alone or withthe addition of a water soluble anti-oxidant. The water soluble oxidantmay be any known biologically effective anti-oxidant, such as GSH,vitamin C, vitamin E and N-acetyl cysteine (NAC).

The present peptides may be prepared according to known pharmaceuticaltechnology. They may be administered singly or in combination, and mayfurther be administered in combination with other cardiovascular drugs.They may be conventionally prepared with excipients and stabilizers insterilized, lyophilized powdered form for injection, or prepared withstabilizers and peptidase inhibitors of oral and gastrointestinalmetabolism for oral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Helical Wheel Projections of therapeutic peptide mimetics asrelated to Apolipoprotein A-I_(Paris) (SEQ ID NO:9) and A-I_(Milano)(SEQ ID NO:1) class A amphipathic alpha helices. Each helix is shownlooking through the barrel of the helix. The dotted line represents thehydrophobic/hydrophilic interface which separates the nonpolar from thepolar face of the amphipathic helix. The cysteine is labeled with anasterisk to note its proximity to the interface.

FIG. 2. Helical Wheel Projection of therapeutic peptide mimetic relatedto Apolipoprotein E3 (SEQ ID NO:47). The helix is shown looking throughthe barrel of the helix. The dotted line represents thehydrophobic/hydrophilic interface which separates the nonpolar from thepolar face of the amphipathic helix. Cysteine112 is labeled with anasterisk to note its proximity to the interface.

FIG. 3. Helical Wheel Projection of synthetic peptide related to theantioxidant domain of Apolipoprotein A-V (SEQ ID NO:52). The helix isshown looking through the barrel of the helix. The dotted linerepresents the hydrophobic/hydrophilic interface which separates thenonpolar from the polar face of the amphipathic helix. Cysteine224 islabeled with an asterisk to note its proximity to the interface.

FIG. 4. Schematic showing protocol for determining the antioxidantactivity of synthetic peptide mimetics which sets criteria for peptidesuseful in the invention. The peptides are first added to a lipoxygenaseassay to observe the rate of lipid peroxidation of micelles (100). Themicelle substrate (100) is composed of1-palmitoyl-linoleoylphosphatidylcholine and dispersed in deoxycholateand Borate (pH=9.0)/saline-EDTA. Second, the peptides are added to anassay having xanthine/xanthine oxidase to observe the rate of lipidperoxidation of micelles (100). Thirdly, the peptides are added to anassay to determine whether the peptides directly quench reactive oxygenspecies and prevent cytochrome reduction.

FIG. 5. Graphs showing antioxidant activity of peptide mimetics of theapoA-I_(Milano) peptide (SEQ ID NO. 1 in FIG. 5A) and the apoA-I_(Paris)peptide (SEQ ID NO: 9 in FIG. 5B). Phospholipid (PL) micelles wereexposed to reactive oxygen species (ROS) generated via xanthine/xanthineoxidase (X/Xo, 20 U/ml) in the absence (squares) or presence ofincreasing concentration of the synthetic 18-mers: diamonds, circles,triangles and hatched squares denote 100, 200, 300, and 400 μg/mLconcentrations. Results show that the peptides exhibit antioxidantactivity in dose-dependent manner. Results show that the peptidesexhibit antioxidant activity in dose-dependent manner whereapproximately 50% protection against lipid peroxidation is observedusing 200 μg/ml.

FIG. 6. Graphs showing antioxidant activity of apoA-I_(Milano) peptide167-R173C-184 inhibits oxidation induced via ROS. In FIG. 6A, PLmicelles were exposed to xanthine/xanthine oxidase (X/Xo, 20 U/ml) inthe presence of increasing concentrations of a cysteine-free controlpeptide (167-184, SEQ ID NO: 2) show no difference in rate of oxidationof lipids as compared to no peptides. FIG. 6B shows results using thethiol-containing apoA-I_(Milano) peptide (167-R173C-184, SEQ ID NO: 1)which show increased antioxidation in a dose-dependent manner. Symbolsand doses are the same as in FIG. 5 for Panels A and B. FIG. 6C showsreduction of cytochrome C assay. The synthetic peptides failed toprotect cytochrome C indicating that the thiol-containing peptide basedon apoA-I_(Milano) is unable to directly quench ROS in the aqueousphase. SOD=superoxide dismutase control.

FIG. 7. Biological activities of synthetic apoA-I_(Milano) peptide167-R173C-184 (SEQ ID NO: 1) and synthetic apoA-I_(Paris) peptide145-R151C-162 (SEQ ID NO: 9). FIG. 7A: Interaction of peptide167-R173C-184 with GSH. The combination of GSH plus 167-R173C-184(triangles) inhibits initial rates of lipoxygenase-mediated lipidperoxidation compared to GSH alone (diamonds) and the apoA-I_(Milano)peptide alone (circles). FIG. 7B: Peptide apoA-I_(Paris) 145-R151C-162(SEQ ID NO: 9) can stimulate LCAT activation while peptideapoA-I_(Milano) 167-R173C-184 (SEQ ID NO: 1) failed. Results areexpressed as a percentage of activation obtained with apoA-I_(WT). FIG.7C: Peptides unable to stimulate cholesterol efflux from J774macrophages.

FIG. 8. Graphs show antioxidant activity of a cysteine containingpeptide related to Helix 10,of apoA-I (220-K226C-237, SEQ ID NO: 16).FIG. 8A shows that the thiol containing peptide based on helix 10 ofapoA-I inhibits lipoxygenase mediated oxidation of phospholipid in adose dependent manner similar to the inhibition obtained in FIG. 8Busing the 167-R173C-184 peptide. Symbols and doses are the same as inFIG. 5.

FIG. 9. Graphs showing antioxidant activity of a synthetic peptide basedon helix 3 of apolipoprotein E-3 (105-R112C-122, SEQ ID NO: 47). In FIG.9A, PL micelles exposed to xanthine/xanthine oxidase (X/Xo, 20 U/ml), inthe presence of increasing concentrations of a cysteine-free peptiderelated to the apolipoprotein E4 (apoE4) isoform (105-122, SEQ ID NO:48), show no difference in rate of oxidation of lipids from the absenceof peptides. FIG. 9B shows results using the thiol-containing peptide(105-R112C-122, SEQ ID NO: 47) that increasing the peptide concentrationinhibits oxidation of phospholipid in a dose-dependent manner where 50%protection is observed at 200 μg/ml. Symbols and doses are the same asin FIG. 5. FIG. 9C shows reduction of cytochrome C (no phospholopids)with X/Xo (squares); triangles X/Xo plus the apoE4 peptide (SEQ ID NO:48) (400 μg/ml); circles, X/Xo plus the apoE3 peptide (SEQ ID NO: 47)(400 μg/ml). Note the synthetic peptides failed to protect cytochrome Cindicating that the thiol-containing peptide (SEQ ID NO: 47) was unableto directly quench ROS in the aqueous phase. The asterisks denote thecontrol SOD (superoxide dismutase).

FIG. 10. Representation of the general placement of a thiol-bearingresidue in peptide mimetics. FIGS. 10A and 10B show the ideal placementof the cysteine residue in an amphipathic alpha helix peptide is at theinterface. FIGS. 10C and 10D show alternate positions of the cysteine tobe in the hydrophobic or the hydrophilic face.

FIG. 11. Representation of the general placement of multiplethiol-bearing residues in synthetic peptides. Cysteine residues can beplaced on the same side of the interface as in FIGS. 11A, 11B, and 11D,or on opposite sides of the amphipathic interface as in FIGS. 11C, 11Eand 11F.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

The term “peptide” herein is used to describe an amino acid sequencebetween 2 and 100 amino acids in length, the amino acids being joined bypeptide linkages. The amino acids may be naturally and non-naturallyoccurring.

The term “antioxidant” herein refers to any compound, composition,peptide or protein that inhibits oxidation of phospholipid. A “potent”antioxidant will inhibit oxidation at an effective concentration (EC50)that produces 50% reduction in phospholipid oxidation.

The term “amphipathic” herein refers to a domain which has both ahydrophobic and a hydrophilic surface that are identified, e.g., asdescribed in Kaiser and Kezdy (Ann. Rev. Biophys. Biophys. Chem. 16:561, 1987; Science 223:249, 1984. The term “amphipathic” further meansthat peptides must exhibit “sidedness” and be amphipathic along the axisthrough the helix, wherein the majority of the residues on the nonpolar,hydrophobic side of the helix are nonpolar residues, preferably leucine,but may include alanine, valine, isoleucine, proline, phenylalanine,tryptophan and methionine. A majority of the residues on the lipophilicside is preferably made up of hydrophilic residues glycine, serine,threonine, cysteine, tyrosine, asparagines, glutamine, aspartate,glutamate, lysine, arginine and histadine.

The term “homology” or “homologous” means an amino acid similaritymeasured by the program, BLAST (Altschul et al (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402), as found athttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi and expressed as −(%identity n/n). In measuring homology between a peptide and a protein ofgreater size, homology is measured only in the corresponding region;that is, the protein is regarded as only having the same general lengthas the peptide, allowing for gaps and insertions.

The terms “derived from” or “based on” mean, regarding a peptide aminoacid sequence, having a relationship to a native sequence of anHDL-associated protein.

The term “substantially identical” is herein used to mean having anamino acid sequence which differs only by conservative amino acidsubstitutions or by non-conservative amino acid substitutions,deletions, or insertions located at positions which do not destroy thebiological activity of the peptide.

The term “HDL-associated protein” or “HDL-related protein” means aprotein and/or apolipoprotein that is naturally associated with HighDensity Lipoproteins (HDL), derived from either plasma or interstitialfluids that can be isolated within the HDL density interval (i.e.d=1.063-1.25 g/ml fraction) of co-isolates with apoA-I uponimmunoaffinity procedures. Moreover, the said apolipoproteins may alsobe present in lipid-free form and participate in HDL metabolic pathwaysincluding the ABCA1 cholesterol efflux pathway which gives rise to HDLparticles.

The term “Class A amphipathic alpha helix” refers to an alpha helix inwhich one surface of the peptides is composed primarily of hydrophobicamino acids and the other surface hydrophilic amino acids. Class A alphahelices possess positively charged amino acids on the polar surface nextto the interface of the hydrophobic domain, and negatively chargedresidues in the middle of the polar surface.

The term “Class Y alpha helices” refers to an alpha helices which areamphipathic, exhibiting a broad nonpolar surface and a hydrophilicdomain that lacks interfacial positively charged residues.

Introduction

Cardiovascular disease is the number one cause of death in westernsocieties and the prevalence of this disease is increasing worldwide.One of the strongest predictors of risk is the plasma concentration ofhigh-density lipoprotein (HDL) which exhibits an inverse relationship.Despite the strong epidemiological data relating increased plasma HDL toprotection from cardiovascular disease, a number of rare mutations inapolipoprotein A-I, the major protein of HDL, present an HDL deficiencyand resistance to cardiovascular disease. Apolipoprotein A-I_(Milano)(apoA-I_(Milano)) and Apolipoprotein A-I_(Paris) (apoA-I_(Paris)) arerare, naturally occurring Arg→Cys substitutions in apoA-I primarysequence that manifest such a HDL deficiency, but affected subjects donot develop cardiovascular disease. The cysteine mutations enableapolipoprotein dimerization via a disulfide bridge. This dimerizationlimits HDL particle growth and facilitates the clearance of HDL from thecirculation. Indeed, human carriers of apoA-I_(Milano) exhibit a HDLdeficiency and mild triglyceridemia. The paradox of HDL deficiency andprotection from cardiovascular disease has led to the suggestion thatthe cysteine substitution for arginine in the lipid-binding domain ofapoAI may impart redeeming qualities protecting the artery wall fromathermatous lesion formation.

The inventor reported, for the first time, in Biochemistry 41, 2089-2096(2002), a unique antiatherogenic function of apoA-I_(Milano) andapoA-I_(Paris) related to antioxidant properties on phospholipidsurfaces. The results of the studies indicate that apoA-I_(Milano) andapoA-I_(Paris), were potent inhibitors of lipid peroxidation protectingphospholipid surfaces from lipophilic, as well as, water soluble freeradical initiators; whereas, apoA-I_(WT) was a relatively poor inhibitorof oxidative events.

Using purified recombinant apolipoproteins and enzymatic methods ofphospholipid peroxidation it was demonstrated that apoA-I_(Milano)(R173C) and apoA-I_(Paris) (R151C) exhibited antioxidant activities notassociated with wild-type apoA-I. This antioxidant activity wasattributed to the monomeric form of apoAI_(Milano) and apoA-I_(Paris)and was found to be dependent on the presence of phospholipid. Thelatter is based on studies where apoA-I_(Milano) and a synthetic peptidemimetic were unable to prevent superoxide anion induced reduction inCytochrome C (FIG. 6C).

This observation has important implications regarding the underlyingmechanism by which cysteine containing amphipathic alpha helices in theexchangeable apolipoproteins exert antioxidant activity and protectagainst inflammatory related disease. Xanthine/xanthine oxidasegenerates superoxide anion and hydroxyl radicals in the aqueous phasethat mediate the oxidation of phospholipid. The fact thatapoA-I_(Milano) was unable to prevent xanthine/xanthine oxidase mediatedreduction of cytochrome C suggests that in lipid-free formapoA-I_(Milano) was unable to quench reactive oxygen species inlipid-free form. This indicates that apoA-I_(Milano) (and its syntheticpeptide mimetics) act on the phospholipid to inhibit theinitiation/amphification of lipid peroxidation via a mechanism probablyrelated to chain-breaking antioxidant activity. By extension of theseobservations, one can infer that antioxidant activity is directed towardlipid surfaces which links the antioxidant activity of apoA-I_(Milano)to the ABCA1 transporter that is required for the lipidation ofapolipoproteins in vivo. This provides a basis for drug development inwhich synthetic peptides can be engineered to possess both thelipidation properties of the native apolipoproteins and the novelantioxidant activity discovered for apoA-I_(Milano). As a result, it isfeasible to specifically target the therapeutics to sites ofinflammation and cholesterol deposition where there is an upregulationin the ABCA1 transporter to specifically deliver potent antioxidantactivity to sites where its needed most to prevent inflammatory relateddisease initiation.

The gene for human Apolipoprotein A-I (Apo A-I) had been previouslycloned. The entire sequence for the human Apo A-I protein is found atSEQ ID NO:85. Certain mutations in this gene have been identified at themolecular level, such as Apolipoprotein A-I_(Milano) (Apo A-I_(Milano))(R173C) and Apolipoprotein A-I_(Paris) (Apo A-I_(Paris)) (R151C).

Using the observation that a free thiol positioned at thehydrophobic/hydrophilic interface of an amphipathic alpha helix conferschain breaking antioxidant activity associated with an inhibition inlipid peroxide amplification on phospholipid surfaces, permits thedesign and synthesis of peptides that have anti-oxidant activity andwhich thereby allow prevention of inflammatory events associated withthe onset of CVD and other diseases.

Designing peptides that exhibit antioxidant activity that are activeonly upon lipidation in effect enables these peptides to be targeted toareas where there is inflammation and cholesterol deposits. Thesynthetic peptides based on apoA-I_(Milano) and apoA-I_(Paris) bind tolipid surfaces and exert the antioxidant characteristics of thefull-length variants. These specific peptides do not promote cholesteroland phospholipid efflux from cells indicating that they are useful asanti-inflammatory agents directed towards preformed HDL and metabolicpathways linked to HDL metabolism. Utilizing information based on theposition of the cysteine residue at the polar/nonpolar interface ofamphipathic alpha helices, it is possible to create peptides derivedfrom different amphipathic alpha helices of apoA-I which are known toexert cholesterol efflux properties. These specific sequences include,but are not limited to helix 1 (aa 44-65) and helix 10 (aa 209-238)which do mediate the lipidation process establishing a specific link tothe ABCA1 transporter. This acts as an entrance to the pathway wherebythe ABCA1 receptor, which is responsible for HDL assembly in the arterywall, is upregulated upon cholesterol enrichment of cells. As a result,it is possible to couple the lipidation properties of native apoA-I withthe phospholipid directed antixodant activity of apoA-I_(Milano) in theform of a synthetic peptide that specifically targets metabolicallyactive sites of cholesterol deposition thus inhibiting inflammatoryevents involved in early disease progression.

Structural analyses revealed identical placement of a cysteine residueat the polar/nonpolar interface of an amphipathic alpha helix withinapoE3 thus defining the mechanism for the antioxidant activity of apoE3.A similar “motif” in apoAV is also used to create new peptides. Thus thecurrent peptides take advantage of a unifying structural domain thatconfers a newly discovered beneficial activity to a broad spectrum ofapolipoproteins that exhibit diverse anti-inflammatory activities. Thisnovel feature of specific cysteine placement within amphipathic alphahelices thus provides the basis for the development of therapeuticagents that have wide applicability to prevent the onset of a number ofinflammatory related diseases including atherosclerosis, Alzheimer'sdisease, and osteoporosis.

Moreover, these peptides have been designed to have both nativeproperties of HDL-associated proteins and the newly discoveredantioxidant property. Because the present peptides are based onnaturally occurring proteins, they are expected to have above averagesafety and efficacy profiles.

Additional peptides are derived from different amphipathic alpha helicalrepeats of apoA-I including Class Y helices, and combinations thereof,to create novel peptides that possess the native properties of apoA-I inpromoting cellular cholesterol efflux in addition to the novelantioxidant activity of apoA-I_(Milano). This will effectively targetpeptides to sites of cholesterol deposition and inflammation where thereis an upregulation in ABCA1 cholesterol transporter expression. (TheABCA1 transporter is a recently discovered HDL receptor located onaortic macrophages associated with Tangier's Disease and is responsiblefor the synthesis of HDL in the artery wall.) This is made possible bythe unique antioxidant property of the synthetic peptides which isconferred upon lipidation on phospholipid surfaces as well as theability to incorporate a free cysteine residue within different classesof amphipathic alpha helices. As a result the peptides can be used incombinations with other therapeutic regiments that promote anunregulation in ABCA1 to effectively target to therapeutic peptides tosites of inflammation and cholesterol deposition

A. Basic Peptide Mimetic Features

The helical wheel projections in FIG. 1 indicate the position of R173Cand R151C within amphipathic alpha helices 6 (residues 145-166) and 7(residues 167-188) of apoA-I. FIGS. 2 and 3 indicate similar positioningof R112C and C224 in amphipathic alpha helices of apoE3 and apoAV,respectively. Each helix is shown looking down the barrel of the helix.The hydrophobic/hydrophilic interface is drawn as the dotted line toseparate the polar from the nonpolar face of the amiphipathic helix. Thenonpolar face is the helix is the side and site of lipid interaction,while the polar face interacts with water and water-soluble free radicalinitiators.

The cysteine substitutions for arginine in FIG. 1 correspond toapoA-I_(Milano) (helix 7) and apoA-I_(Paris) (helix 6). Thesubstitutions are located near the interface of thehydrophobic/hydrophilic surfaces of their respective amphipathic alphahelices. This unique location of the cysteine residue near thehydrophobic/hydrophilic interface demonstrate that cysteinesubstitutions at strategic loci on the amphipathic alpha helices ofHDL-associated proteins can protect phospholipids from water solublefree radical initiators in addition to lipoxygenase-mediated mechanismswhich occur on phospholipid surfaces. Moreover, the apparent wateraccessibility of the free thiol allows for important interactions withwater-soluble antioxidants enhancing the antioxidant activity ofapoA-I_(Milano) and its peptide mimetics.

Based on this model, the peptides of this invention take advantage ofthis observation and direct the design of peptides that have a cysteineresidue present at the polar/nonpolar interface of an amphipathic alphahelix. The generated peptide should thereby exhibit an antioxidantproperty which can thus protect phospholipids from water soluble freeradical initiators.

Since a goal of this invention is to form peptide mimetics that arederived from naturally occurring proteins, so as to be safe and notprone to eliciting an immune response in a patient, it is preferred thatthe present peptides are derived from HDL-associated proteins.Appropriate HDL-associated proteins include but are not limited to,Apolipoprotein A-I, Apolipoprotein A-V, Apoliproprotein E3,Apolipoprotein E4, Human Serum Paraoxonase and their variants. As eachof these proteins is associated with HDL and exhibit the same apparentstructural motif, the current peptides of this invention are derivedfrom specific amphipathic alpha helices in these proteins that known topossess cysteine residues and/or derived from helical segmentsengineered to possess a free cysteine at the polar/nonpolar interface ofamphipathic alpha helices.

It is also important that the peptides that are created meet with thefollowing criteria, that they 1) exhibit antioxidant activity that isdirected toward lipid surfaces, 2) be unable to quench water-solublefree radicals in the absence of lipids and 3) have potentialinteractions with water-soluble antioxidants. These peptides can betested to meet this criteria through several simple experiments whichtake minutes to complete. FIG. 4 outlines these three simple assays usedto define that antioxidant characteristics of each peptide. The assay totest antioxidant activity directed toward lipid surfaces can be testusing the assay described in Example 3. The assay in Example 4 can beused to test potential interactions with other water-solubleantioxidants and the Cytochome C reduction assay. Example 7 can be usedto test whether the peptide is unable to quench water-soluble freeradicals absent lipids. Other assays that are disclosed in Examples 4and 5, which test whether the peptide is capable of LCAT activation orcholesterol efflux from cells, are used to determine whether thepeptides exhibit the native properties of the protein from which thepeptides is based on.

Preferably these peptides should exhibit a percent protection ofphospholipids from oxidation at a concentration that is at least thesame level of the apoA-I Milano and Paris variants as shown in FIG. 5.Therefore, suitable peptides should exhibit at least a 50% protectionagainst maximum accumulation of lipid peroxides at a concentration ofpreferably no more than 200 μg/mL. The peptides should more preferablyexhibit 50% protection of maximum accumulation of phospholipids at a 250μg/mL concentration, even more preferably no more than 300 μg/mL, andeven more preferably no more than 400 μg/mL of peptide, and mostpreferably at concentrations no greater than 500 μg/mL.

The peptides may be made and purified by methods known in the art,preferably by in vitro automated synthesis, but also by recombinant DNAmethods. Furthermore, these peptides can be synthesized using L-aminoacids, non-natural or other modified amino acids, as is known in theart, in order to synthesize peptides which can act upon targets in thebody and be degraded, yet do not interfere with normal protein function.The peptides can be stored in lyopholized form and dissolved in aqueousbuffers or water prior to use. For the purposes of experimental use, thepeptides are dissolved in sterilized degassed buffers to optimizebiological activity which remains stable over 1-3 months at 4° C.

The synthetic peptides of the present invention could contain at least1-4 cysteine residues. In place of cysteine, other residues may beemployed that also contain a reducing moiety, namely a thiol (SH) group.Creating peptides with other thiol-bearing moieties would confer anincreased nucleophilicity and thereby increase the ability to reducefree radicals. However, this could potentially interfere with importantinteractions with water-soluble antioxidants and thus limit potentialuse in humans.

B. Designing Peptides from HDL-Associated Proteins

The starting point for the present model is Apolipoprotein A-I and otherHDL-associated proteins. As shown in the sub-sequences in Examples 7-13,the present peptides are derived from several regions of the wild-typeApo A-I protein (SEQ ID NO:85), Apolipoprotein E3 (apoE3) (SEQ IDNO:86), Apolipoprotein A-V (apoA-V) (SEQ ID NO:87) (Science 294:169-173)and Human Serum Paraoxonase (PON) (SEQ ID NO:88), particularly regionsthat have amphipathic alpha helices that contain a cysteine residue atthe polar/nonpolar interface.

Good candidates for peptides useful in the invention are peptides basedon protein domains of HDL-associated protein molecules that areamphipathic alpha helices having a cysteine at the hydrophic/hydrophilicinterface because such regions are most likely to interact with lipidsurfaces and be able to confer antioxidant activity. Generally, thehelical segments are marked at their boundary by proline residues.Helical wheel projections as shown in FIG. 1-3 are constructed to definethe position of the cysteine residue at the polar/nonpolar interface ofthe helix. Additional helical segments not known to possess cysteineresidues are used because their unique qualities that permit lipidation(i.e. allow the peptides to promote cholesterol efflux from cells suchas J774 macrophages). Important helical segments are identified based onpublished information (J. Biol. Che., 274: 2021-2028) include helix 1(aa 44-65) and helix 10 (aa 209-238) of apoA-I that are important in thenucleation of lipidation.

C. Cysteine Placement that Influences Antioxidant Activity

Peptides based on helix 1 and helix 10 of apoA-I can be used in the formof a single 18-mer in which a cysteine residue has been strategicallyadded to the polar/nonpolar interface of the amphipathic alpha helix asin apoA-I_(Milano). The position of the cysteine residue is set between1 and 4 amino acids off the interface to mimic the position in thenatural variants. In an idealized model peptide (18-mer) in which thenumbering represents a consecutive sequence of amino acids 1-18, thecysteine can be placed at positions 7, 11, 18 or 5, 9, or 16 dependingon the positioning of the nonpolar face of the helix in the nativestructures defined by the helices derived from apolipoproteins withknown cysteine residues such as apoA-I_(Milano) and apoA-I_(Paris).However, this generalized numbering scheme may not apply to all helicessuch as helix 1 of apoA-I (aa 44-61) where the cysteine can be placed atpositions 4, 11, 18 or 2, 6, 13 to mimic the natural positioning of thecysteine residue at the interface of amphipathic alpha helices. But, ingeneralized terms, the cysteine residue can be placed between 1-4residues off the interface of the helix to confer thiol dependentantioxidant activity.

The ability to utilize amphipathic alpha helices that are riot known topossess a cysteine residue, such as helix 1 and 10 of apoA-I, is basedon the following observations: 1) the synthetic peptides derived fromhelix 6 and 7 of apoA-I_(Milano) and apoA-I_(Paris) (peptides SEQ ID NO:1 and SEQ ID NO: 9, respectively) as well as the peptide derived fromapoE3 all possess the same antioxidant characteristics despite the factthat they differ in primary amino acid sequence (data shown in FIGS. 5,6 and 9). This highlights the critical contribution of specific cysteineplacement which is conversed in each of the parent apolipoproteins. 2) Acysteine residue added to the polar/nonpolar interface of helix 10 ofapoA-I (peptide SEQ ID NO: 16) possesses antioxidant activity similar tothe peptide derived from apoA-I_(Milano) (data shown in Example 8 andFIG. 8).

Specific placements of a free cysteine residue render the amphipathicpeptide relatively protected against free radical mediated oxidation ofphospholipid. The free thiol group does not directly quench the freeradical, but instead prevents the initiation/amphification of lipidperoxidation.

There are three categories that represent specific placement of cysteineresidues around the face of an amphipathic alpha helix as shown in thehelical wheel projection of an 18-mer peptide in FIG. 1-3. First,placement of a cysteine residue at the polar/nonpolar interface of thehelix (i.e. 1-3 amino acids away from the interface as judged by the twodimensional face of the wheel projection) renders the peptide fullyeffective as an antioxidant when oxidation is initiated in the waterphase or on the phospholipid surface. Utilizing a generalized numberscheme for the sequence of amino acids (1-18) in an 18 mer peptide,peptides can be engineered from the following apolipoproteins where theposition of the cysteine residues in noted by numeric position:apoA-I_(Milano) and apoA-I_(Paris) based peptides, positions 7, 11, 18or 5, 9, 16; ApoE3 based peptides, positions 4, 8, 15 or 3, 7, 10; apoAVbased peptides, positions 4, 11, 15 or 6, 13, 17; generic peptide I(18-A) based peptides, 4, 8, 15 or 2, 9, 13; and the generic sequenceII, positions 7, 11, 18 or 5, 9, 16.

Second, placing the cysteine residue in middle of the hydrophobic orhydrophilic face of the helix may impart functionality or result in lossof functionality by disrupting salt bridges, etc., depending on what thegoal of the peptide use is.

Third, peptides with multiple cysteines may be made by placing cysteinesat the hydrophobic/hydrophilic interface, as well as in either thehydrophobic or hydrophilic faces of the helix. Such changes in placementof the cysteine residue is predicted to specifically alter thefunctionality of the peptides in a systematic fashion. This can lead tothe creation of a generic amphipathic alpha helix for the development oftailor-made pharmacteuticals of defined functionality including specificantioxidant activity attributed to strategic cysteine placement, LCATactivation properties endowed via arginine clustering at thepolar/nonpolar interface, and cholesterol efflux properties obtained byeither phenylalanine placement or by combining unique helical segments.

The generalized placement of a cysteine at the polar/nonpolar interfaceof amphipathic alpha helix is represented in FIGS. 10 and 11. FIGS. 1-3also illustrate the conservation of the cysteine placement inapoA-I_(Milano), apoA-I_(Paris), apoE3 and apoAV. In some instances thespecific cysteine placement disrupts salt bridges which may be importantfor allowing increased mobility of the free thiol at thehydrophobic/hydrophilic interface. This is true for peptides based onapoA-I_(Milano), apoA-I_(Paris) and apoE3 which result from R→Cinterchanges in class A amphipathihc alpha helices.

By definition, class A amphipathic alpha helices possess positivelycharged amino acids such as arginine (R) at the interface of thepolar/nonpolar surface of the helix which may be important in designingtherapeutic peptides. In general, the positioning of the cysteine ispositioned near the interface either 1-3 or 1-4 amino acids off theinterface into the aqueous phase which may be utilized to generate apeptide with antioxidant activity. Antioxidant activity of peptides,wherein the cysteine is placed further away from the nonpolar surface,may be influenced by the overall lipid binding affinity of the helicalsegment and its ability to penetrate into phospholipid surfaces. Lipidbinding affinity is influenced by the contribution of specifichydrophobic amino acids located in the nonpolar face of the helix aswell as the distribution of positively charged residues such as lysineand arginine residue in the polar surface promote electrostaticinteractions with phospholipids.

An important feature of some of the synthetic peptides is the movementof the cysteine to the middle of the nonpolar surface of the helixwhich, in and of itself, may not influence antioxidant activity of theindividual synthetic peptide. But such peptides are theorized to lackspecific interactions with water-soluble antioxidants that enhanceantioxidant activity of the thiol-containing apolipoproteins, such asapoA-I_(Milano), and their peptide mimetics. Loss of such importantinteractions would generate an important series of peptide mimetics thatcould be used experimentally to determine whether such interactions areimportant in preventing inflammatory related diseases, thereby allowingthe identification of new drug targets and permitting future drugdesign.

In general, the free cysteine residue can be moved around the face ofthe helix in single-turn fashion as illustrated in the peptide based onapoA-I_(Milano). The natural position of the cysteine residue inapoA-I_(Milano) (helix 7, aa 167-184) is found at position 173. Movementof the thiol around the face of the helix is achieved via specificplacement at 171 (located at the opposite interface), 172 (thiolpositioned in the middle of the hydrophilic surface), and 174 (thiolpositioned in the middle of the hydrophobic surface). However, it ispossible that cysteine placement at other interfacial sites confersantioxidant activity and it is possible that other sites towards themiddle of the hydrophobic surface are useful in designing peptides.

Moreover, various placement schemes are used to create peptidescontaining two or more free cysteine residues. For example, peptides canbe engineered to possess two or three thiols: one in the water face ofthe helix, one in the lipid face, and one at the polar/nonpolarinterface. General examples of the structural placement of multiplecysteine residues are represented in FIG. 11. This last strategy ofcysteine placement is to be used with caution as the multiple cysteinesmay serve to dilute the specificity and unique properties of thepeptides rather than enhance their antioxidant properties.

D. Extending the Peptides

The present peptides are based on a modeled amphipathic alpha helicalstructure. Accordingly, they may be from about 12 to 100 amino acids inlength, preferably 18-40 amino acids in length, more preferably 18-20amino acids in length. The peptide subsequences can be extended ineither the amino and carboxy direction or both, with the sequence fromthe native protein from which the peptide was derived.

When extending the peptides, in one embodiment, beyond the peptideamphipathic helix in the amino and/or carboxy directions, it ispreferred that the sequence of the native Apo A-I, as set forth in SEQID NO:85, is used. In a separate preferred embodiment, the sequences ofnative Apolipoprotein E3 (apoE3) as set forth in SEQ ID NO:86,Apolipoprotein A-V (apoAV) as set forth in SEQ ID NO:87, or human serumparaoxonase (PON) as set forth in SEQ ID NO:88, are used to extend thepeptide.

The extended sequence need not be identical to the recited sequencesabove, however it should be substantially identical, preferably at least80% homologous.

In another preferred embodiment, multiple amphipathic alpha helicalpeptides having cysteine substitutions can be used to extend thepeptides to create a larger peptide in which multiple domains haveantioxidant properties. See Example 8, specifically SEQ ID NOS: 30, 31,39-46.

Depending upon what the targeted disease, proteins and events are willdictate which sequence is used to extend the peptides. For example, ifthe targeted oxidation events are related to Alzheimer's Disease, thenthe peptide should probably be extended with sequence having homology toapoE3. If the targeted oxidation events are related to atherosclerosis,the peptide can be extended with the sequences homologous to apoA-I orPON.

E. Applications and Therapeutics

These peptides make feasible the preparation and administration (eitherorally or intravenously) of agents that carry the beneficial propertiesof the full length apoA-I_(Milano) and apoA-I_(Paris) proteins. Thepresent peptides may be used to prevent CVD in the general population,based on this newly described activity associated with the presence ofthe free thiol in the monomeric form of apoA-I_(Milano) and its peptidemimetics. Therapeutics derived from the dimeric form of the variant,which lacks the antioxidant activity attributed to the monomeric form ofthe variant, is currently in pharmaceutical development. The presentantioxidant peptides are also useful in preventing ischemia followingbypass surgery and/or after myocardial infarction, since the presentpeptides move into and out of artieries with lipoproteins such as HDL. Arecently discovered HDL receptor located on aortic macrophages isassociated with Tangier's Disease (the ABCA1 transporter) and isresponsible for the synthesis of HDL in the artery wall. In oneembodiment, the present peptides may be used to promote cellularcholesterol removal from macrophages in the arterial wall via ABCA1.

The present peptides may be prepared according to known pharmaceuticaltechnology. They may be administered singly or in combination, and mayfurther be administered in combination with other cardiovascular drugs.They may be conventionally prepared with excipients and stabilizers insterilized, lyophilized powdered form for injection, or prepared withstabilizers and peptidase inhibitors of oral and gastrointestinalmetabolism for oral administration.

EXAMPLE 1

Preparation of Synthetic Peptide Mimetics

Synthetic peptides were engineered from the monomeric forms ofapoA-I_(Milano), apoA-I_(Paris) and apoE3. Peptides which lackedcysteine residues were developed from wild-type apoA-I and the apoE4isoform and served as controls. All peptides were purchased fromBiosynthesis Incorporated (Lewisville, Tex.) and were modified by anN-terminal acetyl group and C-terminal amide group. Peptides weredissolved in sterile, filtered, degassed 10 mM Tris-buffered (pH=8.0)Saline EDTA (2.7 mM) and stored at 4° C. The antioxidant activity of thethiol-containing peptides remained stable over a 3 month period whenstored in this manner.

EXAMPLE 2

Micelle Substrate to Test the Antioxidant Activity of the Peptides

A schematic showing the assays used for determining the antioxidantactivity of synthetic peptide mimetics is shown briefly at FIG. 4.Assays 1 and 2 utilize a micelle substrate composed of 1 mM1-palmitoyl-2-linoleoylphosphatidylcholine dispersed in Borate (0.2 M)buffered (pH=9.0)/saline-EDTA (2.7 mM) containing deoxycholate. Thebuffer is made by adding 1.52 grams of deoxycholate to 50 ml of boratebuffer/saline-EDTA. The phospholipid is dried on the surface of a glasstube and resuspended with borate/saline-EDTA/deoxycholate and vortexedto dissolve the lipid. The tube is incubated at 37° C. for 10 minutesand allowed to cool to 25° C. before use.

EXAMPLE 3

Assay to Test Perodixation of Phospholipids in Presence of Peptides

The oxidation system consisted of a micelle substrate composed of1-palmitoyl-2-linoleoylphosphatidycholine (3 mM) dispersed in borate(pH=9.0)/saline-EDTA (2.7 mM) and deoxycholate (6 mM) as described.Phospholipid micelles were used throughout most of these studies tooptimize rates of lipid peroxidation catalyzed by specific enzymes. Thispermitted us to quantify initial rates reliably and in reproduciblefashion. Soybean lipoxygenase (5 U/μl) and xanthine (0.2 mM)/xanthineoxidase (20 U/ml) were used to initiate lipid peroxidation following theaddition of recombinant apolipoproteins to the phospholipid micelles.Increases in conjugated dienes (lipid peroxidation) were monitored byultraviolet absorption spectroscopy (234 nm) at 25° C. The mass ofphospholipid hydroperoxides was calculated using the molar absorptivitycoefficient (ε=29,500 Lcm⁻¹mol-⁻¹) of conjugated dienes. This is madepossible because the phospholipid used possesses only two carbon-carbondouble bonds; as such, only one conjugated diene species is formed perphospholipid molecule. Initial rates of lipoxygenase mediated lipidperoxidation are calculated from the slopes of the linear portion of theoxidation. curves and results can be expressed as nmoles of phospholipidperoxide formed/min.

Based on the maximum levels of lipid peroxide accumulation obtained inthe absence of peptide (i.e. the plateau associated with the oxidationcurves), it is possible to derive quantitative information regarding thepotency of the peptide (i.e. the concentration of peptide resulting in50% protection against lipid peroxidation). Thiol-containing peptidesbased on apoA-I_(Milano), apoA-I_(Paris) and apoE3 generally give 50%protection at a concentration of 200 μg/ml.

EXAMPLE 4

Assays to Test Potential Interaction of Peptides with otherWater-Soluble Anti-Oxidants

Interactions of between apoA-I_(Milano) (and other peptides) withreduced glutathione were evaluated using phospholipid micelles andlipoxygenase (5 U/μl). The latter initiates lipid peroxidation onphospholipid surfaces. Glutathione (GSH) is used a concentration whichrange from 0.025 to 0.1 mM which is added to the phospholipid micellesbefore the addition of lipoxygenase. GSH is also added in combinationwith apoA-I_(Milano) (or its peptide mimetics) and lipid peroxidationmonitored at 234 nm. The capacity of GSH plus apoA-I_(Milano) (or otherpeptides) to inhibit lipid peroxidation is compared to the inhibitoryaction of the thiol-containing compounds alone. Water-soluble freeradicals useful for this assay include but are not limited to any knownbiologically effective antioxidant, such as GSH, vitamin C, vitamin Eand N-acetyl cysteine (NAC).

EXAMPLE 5

Assay to Test LCAT Activation Properties of Synthetic Peptides.

ApoA-1 is a cofactor of LCAT which esterifies cholesterol on HDL. Theability of synthetic peptides to activate Lecithin: CholesterolAcyltransferase (LCAT) was examined using a standard proteoliposomesubstrate (Chen and Alber, J Lipid Res. 23:680-691). The substratecontained the synthetic peptide of interest, phosphatidylcholine (eggyolk PC) and unesterfied cholesterol at the following mole ratios:15:250:12.5. Trace amounts of [14C]cholesterol are added to theproteoliposome during preparation. To monitor cholesterolesterification, reaction mixtures are prepared with the followingconstituents, [14C]cholesterol containing proteoliposomes (4.4×10⁵dpm/ml), 20 mM Tris (pH 8.0), 0.15 mM NaCl, 0.27 mM EDTA, 0.5% humanserum albumin, 2.0 mM β-mercaptoethanol and recombinant human LCATenzyme (20 μg/ml). Results are expressed as a percentage of[14C]cholesterol converted to [14C]cholesterol esters in a 30 minuteassay at 37° C.

EXAMPLE 6

Assay to Determine Cellular Cholesterol Efflux Capability of thePeptides

The main function of ApoA-I is promoting cholesterol efflux from cells.This process results in formation of HDL particles. Therefore this assayis used to show that the peptides possess the native properties ofapoA-I in promoting HDL assembly. The murine macrophage cell-line, J774,was used as cholesterol donors for efflux studies to lipid-freeapolipoproteins or synthetic peptides. This cell-line was chosen becauseit has recently been shown to possess an active apolipoprotein-mediatedefflux pathway involving ABCA1 which is up-regulated by the cAMP analog,8-(4-chlorophenthio)adenosine 3′:5′-cyclic monophosphate. Briefly, 1×10⁵cells/ml were seeded into 24 well culture plates and labeled with 1μCi/ml of [3H]cholesterol dispersed in RMPI 1640 medium containing 1%FBS. Confluent monolayers of radio-labeled cells were equilibrated (2 h)with RPMI containing 0.2% BSA and extensively rinsed with serum-freeRPMI prior to addition of recombinant apolipoproteins or syntheticpeptides. In some instances 0.3 mM of the cAMP analog was added toserum-free medium to upregulate cellular cholesterol efflux. Lipid-freeapolipoproteins and/or synthetic peptides were added (25 μg/ml) toserum-free RPMI and applied to cells. At specified times, aliquots ofmedium were removed and cellular debris pelleted by centrifugation(1000×g, 10 min). Results were expressed as a percentage of the initialcellular [3H]cholesterol appearing in the medium at each time point.

EXAMPLE 7

Assay Conforming that Peptides do not Quench Water Soluble ReactiveOxygen Species

A stock solution (1 mg/ml) of cytochrome C is prepared and 50 μg/ml isadded to 0.2 mM xanthine solution. Xanthine oxidase (20 U/ml) is addedto generate water soluble reactive oxygen species (i.e. superoxide anionand hydroxyl radicals). Reduction of cytochrome C is followed at 550 nmover a time course to determine whether synthetic peptides directlyquench the reactive oxygen species (ROS) in the absence of phospholipid.

The rate of reduction of cytochorme C was compared between X/Xo, X/Xoplus the control peptide (400 μg/ml), X/Xo plus the, thiol-containingpeptide (400 μg/ml) and SOD (superoxide dismutase) was used as acontrol. The synthetic peptides should fail to protect cytochrome Cindicating that the thiol-containing peptide is unable to directlyquench ROS in the aqueous phase.

EXAMPLE 8

Designing Peptides from Amphipathic Helices in ApoA-I_(Milano)

The following lists sequences of amino acids used to prepare peptideswhich exhibited the newly discovered anti-oxidant activity ofapoA-I_(Milano). Control sequences based on amphipathic alpha helicesthat lack cysteine residues are also listed. Alternative positions forthe cysteine residues are listed for each sequence and are importantboth therapeutically and as biological tools to investigate theunderlying basis of inflammatory related diseases.

Synthetic peptides based on the primary amino acid (aa) sequence (aa167-184) where the R173C mutation can be found in apoA-I_(Milano). SEQID NO: 1 mimics the precise location of the cysteine residue inapoA-I_(Milano). SEQ ID NO: 2 is peptide based on wild-type apoA-I whichlacks a cysteine residue. The underlined residues in SEQ ID NO: 2represent alternative positions for the cysteine residue. SEQ ID NOS:3-8 show peptides made with the underlined cysteine substitutions.

SEQ ID NO: 1 SDELRQCLAARLEALKEN 167-R173C-184 SEQ ID NO: 2SDELRQRLAARLEALKEN Control wild type 167-184 SEQ ID NO: 3SDELCQRLAARLEALKEN 167-R171C-184 SEQ ID NO: 4 SDELRCRLAARLEALKEN167-Q172C-184 SEQ ID NO: 5 SDELRQRCAARLEALKEN 167-L174C-184 SEQ ID NO: 6SDELRQRLCARLEALKEN 167-R175C-184 SEQ ID NO: 7 SDELRQRLACRLEALKEN167-A176C-184 SEQ ID NO: 8 SDELRQRLAARLEACKEN 167-L181C-184

Synthetic peptides based on the primary amino acid sequence (145-162)where the R151C mutation can be found in apoA-I_(Paris). The sequence inSEQ ID NO: 9 mimics the precise location of the cysteine residue inapoA-I_(Paris). SEQ ID NO: 10, sequence of control peptide based onwild-type apoA-I which lacks a cysteine residue. The underlined residuesin SEQ ID NO: 10 represent alternative positions for the cysteineresidue. The sequences in SEQ ID NOS: 11-15 are peptides made with theunderlined cysteine substitutions.

SEQ ID NO: 9 GEEMRDCARAHVDALRTH 145-R151C-162 SEQ ID NO: 10GEEMRDRARAHVDALRTH Control wild type 145-162 SEQ ID NO: 11GEEMCDRARAHVDALRTH 145-R149C-162 SEQ ID NO: 12 GEEMRCRARAHVDALRTH145-D150C-162 SEQ ID NO: 13 GEEMRDRCRAHVDALRTH 145-A152C-162 SEQ ID NO:14 GEEMRDRACAHVDALRTH 145-R153C-162 SEQ ID NO: 15 GEEMRDRARACVDALRTH145-H155C-162

Synthetic peptides based on amino acids 220-237 of wild-type apoA-I. SEQID NO: 16 lists a sequence that mimics the position of the cysteineresidue at the polar/nonpolar interface of the amphipathic alpha helixas can be found in the apoA-I_(Milano) based peptides (Line 1). SEQ IDNO: 17 corresponds to a control sequence based on wild-type apoA-I thatlacks a cysteine residue. The underlined residues in SEQ ID NO: 17represent alternative positions for the cysteine residues. The sequencesin SEQ ID NOS: 18-29 are peptides made with the underlined cysteinesubstitutions.

SEQ ID NO: 16 PVLESFCVSFLSALEEYT 220-K226C-237 SEQ ID NO: 17PVLESFKVSFLSALEEYT Control wild type 220-237 SEQ ID NO: 18PVLCSFKVSFLSALEEYT 220-E223C-237 SEQ ID NO: 19 PVLECFKVSFLSALEEYT220-S224C-237 SEQ ID NO: 20 PVLESCKVSFLSALEEYT 220-F225C-237 SEQ ID NO:21 PVLESFKCSFLSALEEYT 220-V227C-237 SEQ ID NO: 22 PVLESFKVCFLSALEEYT220-S228C-237 SEQ ID NO: 23 PVLESFKVSCLSALEEYT 220-F229C-237 SEQ ID NO:24 PVLESFKVSFCSALEEYT 220-L230C-237 SEQ ID NO: 25 PVLESFKVSFLCALEEYT220-S231C-237 SEQ ID NO: 26 PVLESFKVSFLSCLEEYT 220-A232C-237 SEQ ID NO:27 PVLESFKVSFLSALCEYT 220-E234C-237 SEQ ID NO: 28 PVLESFKVSFLSALECYT220-E235C-237 SEQ ID NO: 29 PVLESFKVSFLSALEECT 220-Y236C-237

SEQ ID NO: 30 corresponds to amino acids 209-241 of wild-type apoA-I inwhich a cysteine has been added to the polar/nonpolar interface of theamphipathic alpha helix. This peptide possesses both the nativecholesterol efflux properties of apoA-I (J. Biol. Chem. 274:2021-2028)and has been endowed with thiol dependent antioxidant activity. SEQ IDNO: 31 corresponds to a control peptide that lacks a cysteine residues.The underlined residues represents alternative sites for cysteinesubstitutions either singly or in combination to make new peptides: Thecore sequence is identical to that listed above (SEQ ID NO: 17) and theposition of the cysteine residue follow those listed for SEQ ID NO:18-29).

SEQ ID NO: 30 PALEDLRQGLL PVLESFCVSFLSALEEYT KKLN SEQ ID NO: 31PALEDLRQGLL PVLESFKVSFLSALEEYT KKLN

Synthetic peptides based on amino acids 44-61 of wild-type apoA-I. SEQID NO: 32 lists a sequence of a peptide containing a cysteine residue atthe polar/nonpolar interface of the amphipathic alpha helix just as inapoA-I_(Milano). SEQ ID NO: 33 corresponds to a control sequence basedon wild-type apoA-I that lacks a cysteine residue. The underlinedresidues in SEQ ID NO: 33 represent alternative positions for thecysteine residue. SEQ ID NOS: 34-38 are peptides with the underlinedcysteine substitutions.

SEQ ID NO: 32 LKLCDNWDSVTSTFSKLR 44-L47C-61 SEQ ID NO: 33LKLLDNWDSVTSTFSKLR Control wild type 44-61 SEQ ID NO: 34LCLLDNWDSVTSTFSKLR 44-K45C-61 SEQ ID NO: 35 LKCLDNWDSVTSTFSKLR44-L46C-61 SEQ ID NO: 36 LKLLCNWDSVTSTFSKLR 44-D48C-61 SEQ ID NO: 37LKLLDCWDSVTSTFSKLR 44-N49C-61 SEQ ID NO: 38 LKLLDNWDSVTSTFSCLR44-K59C-61

Synthetic peptides based on a combination of helices (209-220 plus44-65) found in wild-type apoA-I. SEQ ID NO: 39 lists the sequence of apeptide containing a cysteine residue located at the polar/nonpolarinterface of an amphipathic alpha helix just as in apoA-I_(Milano). SEQID NO: 40 corresponds to a control sequence based on wild-type apoA-Ithat lacks cysteine residues. The underlined residues in SEQ ID NO: 40represent alternative positions for the cysteine residue. SEQ ID NOS:41-46 are peptides with those underlined cysteine substitutions.

SEQ ID NO: 39 PALEDLRQGLLP LKLCDNWD 209/44-L47C-61 SVTSTFSKLR SEQ ID NO:40 PALEDLRQGLLP LKLLDNWD Control209/ SVTSTFSKLR 44-61 SEQ ID NO: 41PALEDLCQGLLP LKLLDNWD 209-R215C-220/ SVTSTFSKLR 44-61 SEQ ID NO: 42PALEDLRQGLLP LCLLDNWD 209/44-K45C-61 SVTSTFSKLR SEQ ID NO: 43PALEDLRQGLLP LKCLDNWD 209/44-L46C-61 SVTSTFSKLR SEQ ID NO: 44PALEDLRQGLLP LKLLCNWD 209/44-D48C-61 SVTSTFSKLR SEQ ID NO: 45PALEDLRQGLLP LKLLDCWD 209/44-N49C-61 SVTSTFSKLR SEQ ID NO: 46PALEDLRQGLLP LKLLDNWD 209/44--K59C-61 SVTSTFSCLR

EXAMPLE 9

Amphipathic Antioxidant Peptide Based on Human Apolipoprotein E3

Synthetic peptides based on amino acids (105-122) of apoE3. The sequencein SEQ ID NO: 47 mimics the precise location of the cysteine residue inhuman apoE3. SEQ ID NO: 48 corresponds to a control peptide based on theprimary amino acid sequence of apoE4 which lacks cysteine residues. Theunderlined residues in SEQ ID NO: 48 represent alternative positions forthe cysteine residue. The sequences in SEQ ID NOS: 49-51 are peptideswith those underlined cysteine substitutions.

SEQ ID NO: 47 GADMEDVCGRLVQYRGEV 105-R112C-122 SEQ ID NO: 48GADMEDVRGRLVQYRGEV Control wild type 105-122 SEQ ID NO: 49GADMEDCRGRLVQYRGEV 105-V111C-122 SEQ ID NO: 50 GADMEDVRCRLVQYRGEV105-G113C-122 SEQ ID NO: 51 GADMEDVRGCLVQYRGEV 105-R114C-122

EXAMPLE 10

Amphipathic Antioxidant Peptide Based on Apolipoprotein A-V

Synthetic peptides based on Apolipoprotein A-V. SEQ ID NO: 52 mimics theprecise location of the cysteine residue in human apoAV amino acids219-236. SEQ ID NO: 53 corresponds to a control peptide based on thesame sequence as shown in SEQ ID NO: 52 except the cysteine residue hasbeen replaced with a glycine residue to generate a peptide which lacksthe cysteine. The underlined residues in SEQ ID NO: 53 representalternative positions for the cysteine residue.

SEQ ID NO: 52 ARLSRCVQVLSRKLTLKA 219-G224C-236 SEQ ID NO: 53ARLSRGVQVLSRKLTLKA Control wild type 219-236 SEQ ID NO: 54ARLCRGVQVLSRKLTLKA 219-S222C-236 SEQ ID NO: 55 ARLSCGVQVLSRKLTLKA219-R223C-236 SEQ ID NO: 56 ARLSRGCQVLSRKLTLKA 219-V225C-236 SEQ ID NO:57 ARLSRGVQVLSRKCTLKA 219-L232C-236

SEQ ID NO: 58 lists a sequence of 36 amino acids (219-254) found inapoAV. SEQ ID NO: 59 is a control peptide based on peptide listed in SEQID NO: 58 except the cysteine has been replaced with a glycine residue.The underlined residues in SEQ ID NO: 58 represent alternative positionsfor the cysteine residue. SEQ ID NO: 60-63 are the peptides with theunderlined cysteine substitutions.

SEQ ID NO: 58 ARLSRCVQVLSRKLTLKAKAL 219-G224C-254 HARIQQNLDQLREEL SEQ IDNO: 59 ARLSRGVQVLSRKLTLKAKAL Control HARIQQNLDQLREEL 219-254 SEQ ID NO:60 ARLCRGVQVLSRKLTLKAKAL 219-S222C-254 HARIQQNLDQLREEL SEQ ID NO: 61ARLSCGVQVLSRKLTLKAKAL 219-R223C-254 HARIQQNLDQLREEL SEQ ID NO: 62ARLSRGCQVLSRKLTLKAKAL 219-V225C-254 HARIQQNLDQLREEL SEQ ID NO: 63ARLSRGVQVLSRKCTLKAKAL 219-L232C-254 HARIQQNLDQLREEL

SEQ ID NO: 64 lists a sequence based on amino acids 51-72 of apoAV thathas been engineered to possess a cysteine residue at the polar/nonpolarinterface of the amphipathic alpha helix just as in apoA-I_(Milano). Thecontrol peptide in SEQ ID NO: 65 does not contain a cysteine residue,but the underlined residues correspond to alternative sites for cysteinesubstitutions.

SEQ ID NO: 64 ATLKDSLCQDLNNMNKFLEKLR 51-E58C-72 SEQ ID NO: 65ATLKDSLEQDLNNMNKFLEKLR Control wild type 51–72 SEQ ID NO: 66ATLCDSLEQDLNNMNKFLEKLR 51-K54C-72 SEQ ID NO: 67 ATLKDCLEQDLNNMNKFLEKLR51-S56C-72 SEQ ID NO: 68 ATLKDSCEQDLNNMNKFLEKLR 51-L57C-72 SEQ ID NO: 69ATLKDSLECDLNNMNKFLEKLR 51-Q59C-72 SEQ ID NO: 70 ATLKDSLEQCLNNMNKFLEKLR51-D60C-72

EXAMPLE 11

Peptide Based on Human Serum Paraoxonase

Human serum paraoxonase (PON1A) possesses thiol-dependent antixoidantactivity, however, the domain structure of the enzyme is not welldefined. It has been reported previously that the enzyme can inhibitlipoxygenase mediated lipid peroxidation (Brushia et al, J. Lipid Res.42:951-958) utilizing the protocols set forth in the Examples whichindicate that peptides derived from aspects of paraoxonase secondarystructure may be useful in the design of therapeutic agents. Not shownis a two dimensional wheel projection of the synthetic peptide thatencompasses the antioxidant domain of human serum paraoxonase. Aminoacid residues 276-293 form an amphipathic alpha helix having a cysteinelocated at the interface of the hydrophilic/hydrophobic interface. Belowis a brief list of peptides that possess beneficial potential asantioxidants. The sequences were derived from the basic criteriaestablished in this patent disclosure including specific cysteineplacement within amino acid stretches separated by proline residues.Moreover, the native PON enzyme is an HDL-associated protein thatappears to possess thiol-dependent antioxidant activity directed towardlipid surfaces.

SEQ ID NO: 71 ETGDLWVGCHP SEQ ID NO: 72 ETGDLWVGCHPNGMKIFFYDSEN SEQ IDNO: 73 LKSLDFNTLVDNISVDP ETGDLWVGCHPNGMK IFFYDSEN

EXAMPLE 12

Amphipathic Antioxidant Peptide Based on a Generic Peptide

The sequence of the published (generic) peptide by Segrest et al., inU.S. Pat. No. 4,643,988, DWLKAFYDKVAEKLKEAF (SEQ ID NO:75), which codesfor an alpha helix unrelated to apoA-I, can be modified for purposes ofthis invention. This peptide has been made to model apoA-I amphipathicalpha helices and used it extensively to study apoA-I structure andfunction (Yancey et al. Biochemistry, 1995, vol 34, 7955-7965). Becauseit has been used often to study alpha helices, cysteine residues can beintroduced into this peptide to model antioxidant activity in a genericsequence. SEQ ID NO: 74 is the Segrest peptide with a Cysteine placed atthe interface. Alternate residues of cysteine substitution areunderlined in control peptide SEQ ID NO: 75. SEQ ID NOS: 76-81 arepeptides having those underlined cysteine substitutions.

SEQ ID NO: 74 DWLCAFYDKVAEKLKEAF 18A-K4C SEQ ID NO: 75DWLKAFYDKVAEKLKEAF 18A control SEQ ID NO: 76 DCLKAFYDKVAEKLKEAF 18A-W2CSEQ ID NO: 77 DWCKAFYDKVAEKLKEAF 18A-L3C SEQ ID NO: 78DWLKCFYDKVAEKLKEAF 18A-A5C SEQ ID NO: 79 DWLKACYDKVAEKLKEAF 18A-F6C SEQID NO: 80 DWLKAFYDKCAEKLKEAF 18A-V10C SEQ ID NO: 81 DWLKAFYDKVCEKLKEAF18A-A11C

EXAMPLE 13

Amphipathic Antioxidant Peptide Based on a Generic Peptide II

The following peptides are hypothetical in nature but were engineered topossess unique structural aspects of apoA-I (helix 1, aa 44-65) that maybe important in promoting cellular cholesterol efflux, as well as, acluster of arginine residues based on helix 6 (aa 145-166) that play arole in LCAT activation. Moreover, the antioxidant activity ofapoA-I_(Milano) has been added to the peptide by virtue of the placementof a free cysteine residue at the polar/nonploar interface of the firsthelical segment (18-mer).

The peptide is arranged in a series of two 18-mers separated by aproline residue. The first and second 18-mers contain a non-polar facecomposed entirely of leucine residues. Conservative substitutions inthese domains for isoleucine, phenylalanine, tryptophan, and/ormethionine residues can be made to increase the hydrophobicity of thepeptide to facilitate lipid interactions. The polar face of this first18-mer is modeled from helix 1 of apoA-I and it lacks salt-bridgeinteractions within the peptide and the overall net charge is zero.Cysteine placement at position 7 within the peptide mimics the positionof the thiol at the polar/nonpolar interface of an amphipathic alphahelix as found in apoA-I_(Milano), apoA-I_(Paris) and apoE3. The second18-mer connected in series via a proline residue is nearly an exactmatch to the first 18-mer except arginine residues have been added atpositions 5 and 16 to mirror the precise arrangement of the conservedamino acids within helix 6 (aa 145-166) of apoA-I. The underlined serineresidue can be replaced with a cysteine residue to add antioxidantproperties to the second helical repeat.

The peptide can be used in combined form as shown in SEQ ID NO: 82 or astwo singular 18-mers to separate biological activities. The underlinedcysteine residue in SEQ ID NO: 83, can be replaced with a serine toremove thiol-dependent antioxidant activity. Conversely, thiol-dependentactivity can be added to SEQ ID NO: 84 by replacing the serine with acysteine residue. The unique feature of the peptides is the ability toprecisely add or remove biological activities in a controlled manner togenerate an array of biological tools to probe the complex etiology ofinflammatory related diseases. This may permit the identification ofspecific biological activities that are most important in protectingagainst disease in various genetic models of atherosclerosis thusopening the door for the development of tailor-made pharmaceuticals tocombat a variety of inflammatory diseases.

SEQ ID NO: 82 LEKLNSCLRDRLSALTDTPLEELRDSLRSRLDA LRST SEQ ID NO: 83LEKLNSCLRDRLSALTDT SEQ ID NO: 84 LEELRDSLRSRLDALRST

EXAMPLE 14

Antioxidant Activity of Synthetic Peptide Mimetics of apoA-I_(Milano).

FIG. 5A and 5B show the oxidation of phospholipid in the absence(squares) and presence of synthetic peptide mimetics based onapoA-I_(Milano) and apoA-I_(Paris), respectively. Oxidation ofphospholipid was achieved by exposing phospholipid micelles to reactiveoxygen specieis generated via xanthine/xanthine oxidase. Peptides basedon apoA-I_(Milano) (SEQ ID NO: 1) and apoA-I_(Paris) (SEQ ID NO: 9)inhibited the oxidation of phospholipid in a dose dependent manner wherediamonds, circles, triangles and hatched squares correspond to 100, 200,300 and 400 μg/ml, respectively. Note that 50% protection was achievedwith approximately 200 μg/ml of peptides derived from eitherapoA-I_(Milano) or apoA-I_(Paris).

FIG. 6A shows that a peptide which lacks a cysteine residue (167-184,SEQ ID NO: 2) failed to inhibit oxidation of phospholipid induced byreactive oxygen species. Peptide 167-R173C-184 based on apoA-I_(Milano)effectively inhibited lipid peroxidation (FIG. 6B), but thethiol-containing peptide was unable to directly quench water-solublereactive oxygen species in the aqueous phase as determined using thecytochrome C assay (FIG. 6C). This indicates that the antioxidantactivity of the peptide mimetic of apoA-I_(Milano) (SEQ ID NO: 1) wasdirected toward phospholipid similar to the full-length cysteinevariant.

Phospholipid micelles were exposed to xanthine/xanthine oxidase (X/Xo,20 U/ml) in the absence of peptide (squares, FIGS. 6A & 6B). FIG. 6Ashows the results of a cysteine-free peptide (167-184) where diamonds,circles, triangles, and hatched squares correspond to 100, 200, 300 and400 μg/ml respectively. FIG. 6B shows results using the thiol-containingpeptide (167-R173C-184); doses and symbols are the same as in FIG. 6A.FIG. 6C shows reduction of cytochrome C (no phospholipids) with X/Xo(squares); triangles, X/Xo plus the apoA-I_(Milano) 167-R173C-184peptide (400 μg/ml); circles, X/Xo plus the apoA-I_(Milano) 167-184peptide (400 μg/ml). Note the synthetic peptides failed to protectcytochrome C indicating that the thiol-containing apoA-I_(Milano)peptide is unable to directly quench ROS in the aqueous phase. SOD(superoxide dismutase) was used as a control.

Interaction of apoA-I_(Milano) peptide 167-R173C-184 with GSH is shownin FIG. 7A. Squares show the oxidation of PL-micelles with lipoxygenase;diamonds, circles, and triangles correspond to oxidation in the presenceof GSH alone (100 μM), peptide alone (200 μg/ml) and peptide plus GSH,respectively. FIG. 7A indicates that the apoA-I_(Milano) peptide mimetic(SEQ ID NO: 1) was able to interact synergistically with reduced GSH toinhibit lipoxygenase-mediated lipid peroxidation. In the absence ofthiol compound, lipoxygenase caused a rapid induction of lipidperoxidation (squares). The presence of reduced glutathione (0.1 mM,diamonds) was unable to effectively inhibit lipid peroxidation comparedto incubations with the peptide mimetic of apoA-I_(Milano) (200 μg/ml,circles). However, the combination of glutathione plus the peptide(triangles) provided even greater protection against oxidation comparedto peptide alone.

FIG. 7B desmonstrates LCAT activation using a standard proteoliposomesubstrate composed of peptides:egg-yolk PC:unesterified cholesterol(15:250:12.5 mole ratios). Results are expressed as a percentage ofactivation obtained with apoA-I_(WT). The synthetic peptide based onapoA-I_(Paris) (SEQ ID NO: 9) was able to activate the emzyme LCAT whilethe apoA-I_(Milano) based peptide (SEQ ID NO: 1) failed in this regard(FIG. 7B). This is probably related to the cluster of three positivelycharged arginine residues (149, 153 and 160) associated with helix 6 (aa145-162) of apoA-I that have been shown to play a role in LCATactivation. This series of arginine residues is present in the peptidebased on apoA-I_(Paris) but it is absent in the peptide based onapoA-I_(Milano).

Both peptides based on apoA-I_(Milano) and apoA-I_(Paris) were unable tostimulate cholesterol efflux from J774 macrophages as shown in FIG. 7C.FIG. 7C: Cholesterol efflux from J774 macrophages; squares, serum freemedium (□); circles, apoA-I_(Milano) peptide (167-R173C-184, SEQ IDNO: 1) (∘); triangles (behind squares), apoA-I_(Paris) peptide(145-R151C-162, SEQ ID NO: 9) (Δ); diamonds, apoA-I_(WT) (⋄).

EXAMPLE 15

Antioxidant Activity of Synthetic Peptide Mimetics Based on Helix 10 ofapoA-I.

In FIG. 8A, PL micelles were exposed to lipoxygenase (5 U/μL) in theabsence (squares) and presence of a synthetic peptide (220-E224C-237)based on helix 10 of apoA-I; diamonds, circles and triangles correspondto 100, 200, 300 μg/mL. For comparative purposes, the ability of peptide167-R173C-184 to inhibit lipoxygenase-mediated lipid peroxidation isshown in FIG. 8B. Squares represent oxidation of phospholipid in theabsence of peptide; diamonds, circles, triangles, and half-darkenedsquares correspond to 100, 200, 300, and 400 μg/ml of theapoA-I_(Milano) based peptide, respectively. Note that both peptidesinhibited lipid peroxidation over the same relative dose rangeindicating that incorporation of a cysteine residue within helix 10 ofapoA-I, which is a Class Y amphipathic alpha helix, is able to conferantioxidant activity like the peptide mimetic of apoA-I_(Milano).

EXAMPLE 16

Antioxidant Activity of Synthetic Peptide Mimetics of apoE3

Antioxidant activity of synthetic peptide, GADMEDVCGRLVQYRGEV (SEQ IDNO: 47), based on helix 3 of apolipoprotein E3 (apoE3) is shown in FIG.9. Phospholipid micelles were exposed to xanthine/xanthine oxidase(X/Xo, 20 U/ml) in the absence of peptide (squares, panels A & B). FIG.9A shows the results of a cysteine-free peptide (105-122) based on theapolipoproteinE4 (apoE4) isoform where diamonds, circles, triangles, andhatched squares correspond to 100, 200, 300 and 400 μg/ml. Note thecontrol peptide (105-122, SEQ ID NO: 48) derived from apoE4 did notinhibit oxidation. FIG. 9B shows results using the thiol-containingpeptide (105-R112C-122, SEQ ID NO: 47) based on apoE3; doses and symbolsare the same as in FIG. 9A. In contrast to the peptide based on apoE4,peptide 105-R112C-122 based on apoE3 inhibited oxidation in a dosedependent manner similar to the peptides based on apoA-I_(Milano) andapoA-I_(Paris). FIG. 9C shows reduction of cytochrome C (nophospholopids) with X/Xo (squares); triangles X/Xo plus the apoE4peptide (SEQ ID NO: 48) (400 μg/ml); circles, X/Xo plus the apoE3peptide (SEQ ID NO: 47) (400 μg/ml). Note the synthetic peptides failedto protect cytochrome C indicating that the thiol-containing peptide(SEQ ID NO: 47) was unable to directly quench ROS in the aqueous phase.The asterisks denote the control SOD (superoxide dismutase).

The present examples, methods, procedures, treatments, specificcompounds and molecules are meant to exemplify and illustrate theinvention and should in no way be seen as limiting the scope of theinvention. Any patents or publications mentioned in this specificationare indicative of levels of those skilled in the art to which the patentpertains and are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference. TheSEQUENCE LISTING accompanying this specification is also herebyincorporated by reference in its entirety.

1. A method of making a non-naturally occurring peptide havingantioxidant activity, the method comprising: producing a peptide of upto 100 amino acids in length comprising an amphipathic alpha helixcomprising 18-40 amino acids, wherein the peptide has a cysteinesubstituted for at least one amino acid residue at or near thepolar/nonpolar interface of the alpha helix in comparison to a naturallyoccurring alpha helix, wherein the cysteine substitution confersantioxidant activity to the peptide.
 2. The method of claim 1, whereinthe peptide is derived from an HDL-associated protein.
 3. The method ofclaim 2, wherein the HDL-associated protein is selected from the groupconsisting of: ApoA-I, Apo E3, Apo E4, Apo AV, and paroxonase.
 4. Themethod of claim 1, wherein the alpha helix comprises a sequence selectedfrom the group consisting of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:17,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:53,SEQ ID NO:59, SEQ ID NO:65, and SEQ ID NO:75.
 5. The method of claim 1,wherein at least one of the amino acids at positions 7, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 26, 27, or 28 of SEQ ID NO:31 is substitutedwith a cysteine.
 6. The method of claim 1, wherein at least one of theamino acids at positions 7, 8, 9, or 10 of SEQ ID NO:48 is substitutedwith a cysteine.
 7. The method of claim 1, wherein the peptide comprisesfrom 18 to 40 amino acids.
 8. The method of claim 1, wherein the peptidecomprises from 18 to 20 amino acids.
 9. The method of claim 4, whereinat least one of the amino acids at position 4, 5, 6, 8, 9, 10, 11, 12,13, 15, 16, or 17 of SEQ ID NO:17 is substituted with a cysteine.